Lipopeptide particles as the immunologically active component of CTL inducing vaccines Ikuo Tsunoda a , Alessandro Sette b , Robert S. Fujinami a , Carla Osero b , Jorg Ruppert b , Carol Dahlberg b , Scott Southwood b , Thomas Arrhenius c , Li-Qing Kuang a , Ralph T. Kubo c , Robert W. Chesnut b , Glenn Y. Ishioka b, * a Department of Neurology, University of Utah School of Medicine, 50 North Medical Dr., Salt Lake City, UT 84132, USA b Epimmune Inc., 6555 Nancy Ridge Dr. #200, San Diego, CA 92121, USA c Cytel Corporation, 3525 John Hopkins Ct., San Diego, CA 92121, USA Received 30 January 1998; received in revised form 4 June 1998; accepted 2 July 1998 Abstract Using a bipalmitoylated lipopeptide consisting of an ovalbumin helper T-cell epitope covalently linked to an in¯uenza virus cytotoxic T-lymphocyte (CTL) epitope, we addressed possible factors that may be critical for CTL induction. Antigen processing of lipopeptide appears to be required for T-cell induction since there was virtually no in vitro binding of lipopeptide to puri®ed MHC molecules. A major portion of lipopeptide immunogenicity was due to its particulate nature inasmuch as CTL induction in mice correlated with insoluble lipopeptide constructs, whereas more soluble analogs were signi®cantly less immunogenic. Immunohistological analysis of tissue from immunized animals revealed that lipopeptide migration from the s.c. injection site to the spleen could be detected as early as 1 h after immunization and cell-associated lipopeptide was observed on macrophages and dendritic cells, implicating both cell populations in the processing and presentation of lipopeptide particles to CTLs. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Lipopeptide particles; Cytotoxic T-lymphocytes; Antigen processing 1. Introduction The importance of class I MHC-restricted cytotoxic T-lymphocytes (CTLs) in conferring protection in the host against viral infection is well established [1±4]. Since CTLs recognize short peptide fragments of viral antigen (Ag) presented in association with class I MHC molecules, a possible strategy for the development of CTL-inducing vaccines has been to use peptide epitopes as the immunogen. A particularly successful strategy for enhancing the immunogenicity of CTL epitopes uti- lizes covalent attachment of long-chain lipids to the peptide moiety. In vivo studies in various animal species have shown that lipidation of T- and B-cell epitopes result in induction of strong antibody (Ab), helper T- lymphocyte (Th), and CTL responses [5±9]. Our labora- tory has developed a modular lipopeptide vaccine con- struct containing two palmitic acid (PAM) residues and a peptide unit composed of a Th and CTL peptide epi- tope covalently linked by a trialanine spacer. Bipalmitoylated Th±CTL constructs containing selected immunodominant epitopes gave vigorous and durable CTL responses in mice [10]. Similarly, we observed strong CTL induction in normal human subjects who were vaccinated with a lipopeptide containing a domi- nant hepatitis B virus (HBV), HLA-A2.1-restricted CTL peptide and a universal tetanus toxin Th epitope [10], the magnitude of which was comparable to that induced during acute HBV infection [11]. These ®ndings help to validate the potential use of lipopep- tides for therapeutic and prophylactic treatment of dis- eases involving CTL immunity. Although lipopeptides are potent T-cell immunosti- mulators, the mechanism underlying their in vivo immunogenicity has yet to be adequately explained. In the following report we examine potential factors that Vaccine 17 (1999) 675±685 0264-410X/99/$19.00 # 1999 Elsevier Science Ltd. All rights reserved. PII: S0264-410X(98)00250-3 * Corresponding author. Tel.: +1-619-404-7171; fax: +1-619-404- 7177.